1) Field of the Invention
The present invention relates to the field of DNA transfection of brain cells.
2) Description of Related Art
Gene therapy is used to treat hereditary diseases such as cystic fibrosis and also acquired diseases such as cancers [M. Conese, et al. Journal of Cystic Fibrosis, 2011, 10, S114], but is only as effective as its ability to deliver the therapeutic polynucleotide to a desired location. Vectors for gene delivery may be viral or nonviral. Viral vectors offer highly efficient gene transfer, but unwanted immune stimulation and the potential for mutagenesis have virtually eliminated them from clinical trials [M. L. Edelstein et al., Journal of Gene Medicine 2007, 9, 833; C. E. Thomas et al., Nature Reviews Genetics 2003, 4, 346]. In contrast, nonviral vectors are safe, have low immunogenicity, and are relatively inexpensive [J. F. Guo et al., Biotechnology Advances 2011, 29, 402].
Examples of nonviral vectors include bacteria [C. H. Chang et al., Biotechnology and Bioengineering 2011, 108], cell penetrating peptides [Y. A. Chen et al., Biomaterials 2011, 32, 4174], functionalized gold nanoparticles or carbon nanotubes [C. M. McIntosh, et al., Journal of the American Chemical Society 2001, 123, 7626; G. Ban et al., Chemical Biology & Drug Design 2006, 67, 78; G. Han et al., Bioconjugate Chemistry 2005, 16, 1356; L. Z. Gao et al., Chembiochem 2006, 7, 239], and cationic polymers. Among these nonviral vectors, cationic polymers including polyethyleneimine (PEI) [U. Lungwitz et al., Eur, J. of Pharmaceutics and Biopharmaceutics 2005, 60, 247], poly(l-lysine) (PLL) [U. Lungwitz et al., Eur. J. of Pharmaceutics and Biopharmaceutics 2005, 60, 247; T. L. Kaneshiro et al., Molecular Pharmaceutics 2007, 4, 759], chitosan [K. Corsi et al., Biomaterials 2003, 24, 1255], dendrimers [J. Dennig, Applications in Materials and Life Sciences 2003, 228, 227; H. M. Wu et al., Biomaterials 2011, 32, 1619] and cationic lipids [M. Morille et al., Biomaterials 2008, 29, 3477] have the advantages of being scalable for manufacturing in quantity and having low immunogenicity, the capacity for selective chemical modification and the ability to carry large inserts. Due to its superior transfection efficiency in a broad range of cell types, synthetic PEI has a privileged place among nonviral gene delivery systems. However, the high number of positive charges on PEI and its lack of biodegradability make it toxic in vivo, which has hampered clinical applications [U. Lungwitz, et al., Eur. J. of Pharmaceutics and Biopharmaceutics 2005, 60, 247; T. L. Kaneshiro et al., Molecular Pharmaceutics 2007, 4, 759].
Chitosan, which is obtained by deacetylation of chitin, is a biocompatible and biodegradable linear polymer whose cationic polyelectrolyte nature provides strong electrostatic interaction with negatively charged DNA to form stable complexes that protect the DNA from degradation. However, the transfection efficiency of chitosan is very low and is dependent on its molecular weight, size and percentage of deacetylation [H. L. Jiang et al. Journal of Controlled Release 2007, 117, 273]. The goal of a successful nonviral gene delivery system, therefore, is to achieve therapeutic efficacy while minimizing toxicity [M. Breunig et al., Proceedings of the National Academy of Sciences of the United States of America 2007, 104, 14454]. To develop such a safe and effective delivery vehicle, PEI-grafted chitosan, chitosan-grafted PEI or a chitosan-PEI composite have been tested and shown to have improved transfection efficiency and reduced toxicity compared to PEI alone [Y. L. Lou et al., Journal of Biomedical Materials Research Part A 2009, 88A, 1058; D. Jere et al., International Journal of Pharmaceutics 2009, 378, 194; H. L, Jiang et al., Gene Therapy 2007, 14, 1389; H. L. Jiang et al., Journal of Biomedical Nanotechnology 2007, 3, 377].
For advanced gene therapy, it is desirable to be able to monitor the in vivo gene delivery in real time. Magnetic resonance imaging (MRI) is a powerful clinical imaging technique for diagnosis of a variety of diseases and post-therapy assessment. MRI contrast can be enhanced by the use of positive or negative contrast agents resulting in brighter (T1-weighted) or darker (T2-weighted) images, respectively. Superparamagnetic iron oxide nanoparticles (SPIONs) are T2 contrast agents that are widely used in molecular and cellular imaging applications [P. Zou et al., Molecular Pharmaceutics 2010, 7, 1974; R. Chen et al., International Journal of Nanomedicine 2011, 6, 511]. Recently, PEI-poly(ethylene glycol) (PEG)-chitosan coated SPIONs have been reported for DNA or siRNA delivery and MRI imaging [F. M. Kievit et al., Advanced Functional Materials 2009, 19, 2244; O. Veiseh et al., Biomaterials 2010, 31] and PEG-grafted PEI-complexed SPION for gene delivery and MRI imaging [G. Chen et al., Biomaterials 2009, 30, 1962]. When incorporated into micelles, a SPION has a longer half-life in circulation and improved biocompatibility, and it shows better contrast. SPION polymeric micelles were used successfully as MRI probes and for drug delivery [N. Nasongkla et al., Nano Letters 2006, 6, 2427; X. T. Shuai et al., Journal of Controlled Release 2004, 98, 41; J. S. Guthi et al., Molecular Pharmaceutics 2010, 7, 32; G. B. Hong et al. Biomedical Microdevices 2008, 10, 693], but they have not been tested for gene delivery.
One area in which gene delivery is particularly difficult is in the targeting of brain tissues. The transport of compounds from the blood to target tissues is restricted by biological barriers such as the blood-brain barrier (BBB). Drug delivery to the brain is particularly hampered because of the tight junctions between adjacent endothelial cells of brain capillaries, which form the BBB. However, some lipid soluble substances can penetrate passively across this barrier, whereas hydrophilic and ionic substances (e.g., amino acids) are transported by a specific carrier transport system.
Efforts have been made to enhance transport via the BBB by conjugating drugs with CNS-permeable moieties. For example, attempts have been made in correcting disorders affecting the CNS system by increasing BBB permeability of exogenous biological compounds such as proteins or specific nucleic acid sequences by conjugating them with lipids. However, none of the prior art approaches provide effective targeting to the brain.